Capsid protein mutant capable of improving infection ability of AAV to eye tissues and application of capsid protein mutant

Abstract

The invention belongs to the technical field of biology, and discloses a capsid protein mutant capable of improving the infection ability of AAV to eye tissues and application of the capsid protein mutant. AAVRFDGTERAA and RQYSDAVRAE are inserted into amino acid at the 587 site and amino acid at the 588 site, which are equivalent to that of wild type AAV2 virus capsid protein, so that capsid protein mutants AAV2-NE01 and AAV2-NE02 are obtained. The virus yield of AAV2-NE01 and AAV2-NE02 is 1.96 times and 1.20 times that of AAV2-WT, after intravitreal injection, cells and trabecular meshwork areas of NHP retina GCL, INL to ONL can be well infected, the infection positive rate is high, the fluorescence intensity is high, the penetrating power to the retina is high, and visual cell nucleuses and inner segments can be infected. The AAV vector has a good application prospect and is expected to be developed into the AAV vector for treating eye diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to capsid protein mutants AAV2-NE01 and AAV2-NE02 capable of improving the ability of AAV virus to infect eye tissue of non-human primates (NHP) and applications thereof. Background Art

[0002] The genome of adeno-associated virus (AAV) is a single-stranded DNA fragment of about 4.7kb, contained in a regular icosahedral non-enveloped viral capsid with a diameter of 20nm, and can be divided into three functional regions: two open reading frames (Rep gene, Cap gene) and terminal inverted repeat sequence (ITR). Among them, the open reading frame of the Cap gene encodes three capsid proteins VP1, VP2 and VP3, with molecular weights of 87, 73, and 61kDa respectively, which are the capsid proteins required for assembly into a complete virus, and they play an important role in viral integration, replication and assembly.

[0003] AAV is a delivery vector widely used in gene therapy. Its principle is to replace the sequence between the ITRs of the AAV genome with the target gene sequence through genetic engineering methods, and then deliver it to the target cells through cell infection to achieve the purpose of gene therapy. Based on the characteristics of safety, high efficiency, stability, sustainability, specificity, and low integration of recombinant AAV, AAV has become one of the main delivery methods in the field of gene therapy.

[0004] However, AAV still faces many challenges: First, the production process is complex, expensive, and production capacity is limited; second, high-dose administration may show neurotoxicity and hepatotoxicity, causing safety issues. Third, the ability to infect some tissue cells is weak. For example, it is difficult to infect the posterior retinal cells when administered through the vitreous cavity, and it is difficult to infect central nervous cells when administered through intravenous administration. Modifying and screening the AAV capsid protein to improve its targeting and transfection efficiency to specific tissues in order to achieve a lower and safer dosage is an effective way to solve this problem.

[0005] Due to the existence of the blood-ocular barrier, the eye has a certain immune exemption, local administration is relatively safe, and fundus diseases are mostly single-gene genetic diseases, so ophthalmology has become a hot area for gene therapy. There are two commonly used injection methods in clinical practice: intravitreal injection and subretinal injection. Subretinal injection has a better infection effect, especially for the RPE layer and outer nuclear layer, but this method is more invasive and can easily cause retinal detachment, which may lead to photoreceptor degeneration, visual function damage and glial cell proliferation. Intravitreal injection has a lower surgical risk, but conventional serotypes such as AAV2 and AAV5 can only infect the RGC layer, and have poor infection effects on cones, rods and RPE. Therefore, the development of AAV mutants that can improve the expression ability of the whole eye, or can penetrate the RGC layer and the inner nuclear layer to infect the outer nuclear layer cells and RPE cells, is of great significance to solving the problem of delivery vectors for ophthalmic gene therapy.

[0006] Previous studies have shown that inserting a polypeptide fragment between sites 587 and 588 of Cap can optimize its ability to infect the eye tissue of cynomolgus monkeys. Previous patent applications CN 116836237A and CN 117143203 A disclosed some capsid protein mutants, and data showed that different fragment insertions had different effects. However, whether this method can improve the ability of AAV virus to infect the retinal tissue of NHPs remains to be further studied. Summary of the invention

[0007] The purpose of the present invention is to overcome at least one deficiency of the prior art and to provide a capsid protein mutant and application thereof that improves the ability of AAV to infect eye tissues.

[0008] The technical solution adopted by the present invention is: The first aspect of the present invention provides: a capsid protein mutant that improves the ability of AAV to infect eye tissue, which is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the AAV capsid protein, and the insertion site is homologous to the amino acids 587 and 588 of the wild-type AAV2 virus capsid protein.

[0009] In some examples of capsid protein mutants, the polypeptides AAVRFDGTERAA or RQYSDAVRAE are inserted into the capsid protein of the AAV2 variant, and the insertion sites are homologous to amino acids 587 and 588 of the wild-type AAV2 virus capsid protein.

[0010] In some examples of capsid protein mutants, the polypeptide AAVRFDGTERAA or RQYSDAVRAE is inserted between amino acids 587 and 588 of the wild-type AAV2 capsid protein.

[0011] In some examples of capsid protein mutants, the amino acid sequence of the wild-type AAV2 capsid protein has a Genbank accession number of YP_680426.1.

[0012] The second aspect of the present invention provides: a gene encoding the capsid protein mutant described in the first aspect of the present invention.

[0013] In some cases of genes, codon optimization was performed depending on the expression system.

[0014] The third aspect of the present invention provides: an expression vector, which expresses the capsid protein mutant described in the first aspect of the present invention, or has inserted therein the gene described in the second aspect of the present invention.

[0015] In some examples of expression vectors, the expression vector is a plasmid or a recombinant AAV vector.

[0016] In some examples of expression vectors, the AAV is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

[0017] In some examples of expression vectors, a gene encoding a functional gene product is also included.

[0018] The fourth aspect of the present invention provides: an AAV virus particle, the shell of which is formed by the assembly of capsid proteins VP1, VP2 and VP3 formed by the capsid protein mutant described in the first aspect of the present invention, or by the assembly of capsid proteins VP1, VP2 and VP3 encoded by the gene described in the second aspect of the present invention.

[0019] The fifth aspect of the present invention provides: a composition comprising the expression vector described in the third aspect of the present invention, or the AAV virus particle described in the fourth aspect of the present invention.

[0020] The sixth aspect of the present invention provides: an application of the composition described in the fifth aspect of the present invention, the application comprising: Preparation of gene therapy formulations targeting ocular tissues; Construct transgenic animal models of eye tissue diseases; Preparation of drugs or transgenic vectors targeted to ocular tissues.

[0021] The beneficial effects of the present invention are: The capsid protein mutants of some examples of the present invention, when assembled with other capsid structural protein subunits to obtain a complete AAV capsid protein, can effectively enhance the ability of AAV virus to infect eye tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the yield results of the adeno-associated virus variants prepared by the solution of the present invention in the test example of the present invention and the wild-type adeno-associated virus in the control group.

[0023] Figure 2 This is a diagram showing the retinal infection effect of cynomolgus monkey eye tissue infected with wild-type AAV2, AAV2-NE01 and AAV2-NE02 viruses of the embodiments of the present invention for 3 weeks.

[0024] Figure 3 This is a diagram showing the infection effect of the trabecular meshwork of cynomolgus monkey eye tissues infected with the wild-type, AAV2-NE01 and AAV2-NE02 viruses of the embodiments of the present invention for 3 weeks.

[0025] Figure 4 This is a picture of retinal immunofluorescence staining of cynomolgus monkey eye tissue 3 weeks after AAV2-NE01 infection in an example of the present invention.

[0026] Figure 5 This is a picture of retinal immunofluorescence staining of cynomolgus monkey eye tissue 3 weeks after AAV2-NE02 infection in an example of the present invention.

[0027] Figure 6 It is a Cap587-588 sequence alignment diagram of the wild-type AAV2, AAV2-NE01 and AAV2-NE02 sequences of the embodiments of the present invention. DETAILED DESCRIPTION

[0028] The first aspect of the present invention provides: a capsid protein mutant that improves the ability of AAV to infect eye tissue, which is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the AAV capsid protein, and the insertion site is homologous to the amino acids 587 and 588 of the wild-type AAV2 virus capsid protein.

[0029] In some examples of capsid protein mutants, the polypeptides AAVRFDGTERAA or RQYSDAVRAE are inserted into the capsid protein of the AAV2 variant, and the insertion sites are homologous to amino acids 587 and 588 of the wild-type AAV2 virus capsid protein.

[0030] The homology with the amino acids 587 and 588 of the wild-type AAV2 viral capsid protein can be determined based on existing methods, such as by comparative analysis of the amino acid sequences of AAV capsid proteins of different serotypes.

[0031] In some examples of capsid protein mutants, the polypeptide AAVRFDGTERAA or RQYSDAVRAE is inserted between amino acids 587 and 588 of the wild-type AAV2 capsid protein.

[0032] In some examples of capsid protein mutants, the amino acid sequence of the wild-type AAV2 capsid protein has a Genbank accession number of YP_680426.1.

[0033] The second aspect of the present invention provides: a gene encoding the capsid protein mutant described in the first aspect of the present invention.

[0034] In some cases of genes, codon optimization was performed depending on the expression system.

[0035] The third aspect of the present invention provides: an expression vector, which expresses the capsid protein mutant described in the first aspect of the present invention, or has inserted therein the gene described in the second aspect of the present invention.

[0036] In some examples of expression vectors, the expression vector is a plasmid or a recombinant AAV vector.

[0037] In some examples of expression vectors, the AAV is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

[0038] In some examples of expression vectors, a gene encoding a functional gene product is also included. The gene product preferably acts on eye tissue.

[0039] The fourth aspect of the present invention provides: an AAV virus particle, the shell of which is formed by the assembly of capsid proteins VP1, VP2 and VP3 formed by the capsid protein mutant described in the first aspect of the present invention, or by the assembly of capsid proteins VP1, VP2 and VP3 encoded by the gene described in the second aspect of the present invention.

[0040] The fifth aspect of the present invention provides: a composition comprising the expression vector described in the third aspect of the present invention, or the AAV virus particle described in the fourth aspect of the present invention.

[0041] The sixth aspect of the present invention provides: an application of the composition described in the fifth aspect of the present invention, the application comprising: Preparation of gene therapy formulations targeting ocular tissues; Construct transgenic animal models of eye tissue diseases; Preparation of drugs or transgenic vectors targeted to ocular tissues.

[0042] In some application examples, the animal is a non-human primate mammal, including but not limited to macaques, crab-eating macaques, Yunnan snub-nosed monkeys, flat-topped monkeys, bear monkeys, red-faced monkeys, marmosets, and slow loris.

[0043] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels. Example

[0044] This example is based on the directed evolution of multiple rounds of 10-12 amino acid polypeptide insertion libraries in the past, and the top 30 sequences with the best comprehensive performance of retinal infection ability and yield are screened from NGS sequencing data to provide multiple adeno-associated virus variants AAV2-NE01, AAV2-NE02, AAV2-PN001 to AAV2-PN028. The preparation of the adeno-associated virus variants includes the following steps: 1. Plasmid construction Through GENEWIZ Biotechnology Co., Ltd., nucleic acid sequences corresponding to amino acid sequences such as AAV2-NE01, AAV2-NE02, and AAV2-PN001 to AAV2-PN028 (as shown in Table 1) were synthesized, and the synthesized sequences were cloned into the pAAV2-WT plasmid by seamless cloning, and the mutant sequences shown in Table 1 were inserted into the 587 to 588 sites of the Cap protein to construct the pAAV2-mutant plasmid vector; the ligation products were transformed into the competent Escherichia coli stbl3, single colonies were picked for culture, and the plasmids were extracted for sequencing and enzyme digestion verification, and the mutant plasmid was successfully constructed as identified by agarose gel electrophoresis.

[0045] Table 1. Amino acid sequence information of AAV2 mutants inserted between N587 and R588 of Cap protein

[0046] 2. Packaging of mutant viruses such as AAV2-WT, AAV2-NE01, and AAV2-NE02 pAAV2-WT or AAV2-mutant packaging plasmid, pAAV-CAG-EGFP (or pAAV-CAG-Mcherry) expression plasmid and pHelper auxiliary plasmid were co-transfected into suspended HEK293T cells. After the transfected cells were cultured for 72 hours, the cells were lysed to harvest the virus, and the virus was purified by iodixanol density gradient centrifugation, which mainly included the following steps: A. Cell culture (1) Take out a tube of suspended 293T cells from the liquid nitrogen tank, put it in a 37℃ water bath, shake it quickly to thaw the cells, add it to a centrifuge tube containing 5mL Wayne 293 serum-free medium, centrifuge it at 200×g for 5min, discard the supernatant, resuspend it in 10mL Wayne 293 serum-free medium, place it in a 125mL triangular shake flask, add Wayne 293 serum-free medium to 20mL, and culture it in a shaking incubator at 120rpm, 37℃, and 5% CO2; (2) After 48-72 hours of culture, the cell density reaches 2-3E+6 cells / mL, and the cells can be subcultured; (3) Take out the seed cells to the clean bench, shake well and then pipette about 500 µL of sample, take 20uL of sample and add 20µL of trypan blue to mix, pipette 20µL of the mixture into the cell counting plate, read the data three times on the automatic cell counter, and take the average value as the cell density; (4) Take an appropriate amount of cells, dilute them to 0.6-0.65E+6 cells / mL with fresh Wayne 293 serum-free medium, and culture them in a 500mL triangular shake flask with a culture volume of 25mL per flask. Culture them in a shaking CO2 cell culture incubator at 120rpm, 37°C, and 5% CO2.

[0047] B. Cell transfection (1) Cultivate the above cells for 48 hours, and the cell density reaches 3-3.8E+6 cells / mL, and the cells can be transfected; (2) For each bottle of cells, add packaging plasmid, pHelper, and pAAV-CAG-EGFP (or pAAV-CAG-Mcherry) (100 μg in total) to 4 mL of DMEM medium and mix thoroughly to prepare the "plasmid dilution solution". (3) Take another 1 mL of DMEM medium and add 300 μg of PEI to mix well, which is the "PEI dilution solution"; (4) Pour the "PEI diluent" into the "plasmid diluent", mix quickly, and let stand at room temperature for 25 minutes to form a transfection complex; add the transfection complex dropwise into the cell solution while gently shaking; (5) Place the culture flask in a shaking CO2 cell culture incubator and culture under the following conditions: 120 rpm, 37°C, and 5% CO2.

[0048] C. Virus Harvest (1) After transfection, cells were cultured for 72 hours and virus harvesting was started. (2) Collect the cell suspension in each bottle into a centrifuge bottle, centrifuge at 1000g for 5 min, collect the supernatant into a new centrifuge bottle, add 0.245mL of 50% PEG8000 solution (containing 0.5mol / L NaCl) per mL of supernatant, mix thoroughly, and let stand at 2-8°C overnight; (3) Add an appropriate amount of 0.5% TritonX-100 cell lysis buffer (containing universal nuclease) to the cell pellet, treat at 37°C for 1-2h, then add 1 / 10 volume of 5 mol / L NaCl, mix thoroughly and centrifuge at 4°C and 3000g for 15min, collect the supernatant, which is the "viral crude extract" and temporarily store at 2-8°C; (4) Centrifuge the supernatant after PEG8000 precipitation at 4°C and 3000g for 15 min, discard the supernatant, and resuspend it in the "viral crude extract" in step (3) for later use.

[0049] D. Virus purification 1) Take an Ultra-Clear centrifuge tube, add 0.5mL 60% Iodixanol, 2mL 40% Iodixanol, 1.5mL 25% Iodixanol, 1.5mL 15% Iodixanol, the collected virus suspension, and finally the cell lysate to balance at the bottom; 2) 10°C, 230,000 g, 8°C, 9°C ultracentrifugation for 18 h; 3) Take an ultrafiltration tube and soak the filter membrane with 1 mL of PBS buffer; 4) Use a pipette to carefully extract the middle layer of 40% Iodixanol-60% Iodixanol in the ultracentrifuge tube, avoiding protein, and transfer it to the ultrafiltration tube; 5) Add an appropriate amount of PBS buffer and blow evenly, centrifuge at 4500g for 3-5 min, and repeat this step 5-7 times to remove Iodixanol; 6) Dilute the crude virus product in step 5 to 8 mL and repeat steps 1 to 5.

[0050] 6) Add 1 mL of PBS buffer and pipette for 40-50 times to form a virus suspension, then transfer to an EP tube; 7) Use a 5 mL syringe to draw the virus suspension in the EP tube and filter it through a 0.22 μm filter. Collect 20 μL of the virus liquid as a test sample and dispense it into 100 μL tubes to obtain adeno-associated virus variants.

[0051] Comparative Example This comparative example provides a wild-type adeno-associated virus (AAV2-WT), which differs from the preparation method of the embodiment only in that the sequence of the adeno-associated virus capsid protein adopts the Cap sequence of the wild-type AAV2 (protein ID: YP_680426.1 SEQID NO.1).

[0052] Test Case 1. Virus titer detection (1) Virus lysis 1) Take 20 μL of the virus samples of Example 1 and Comparative Example respectively; 2) Add 1 μL each of 10% SDS, 0.5 mol / L EDTA, and proteinase K and mix well; 3) Incubate at 56°C for 1 hour in a thermomixer, then at 90°C for 10 minutes; 4) Take 10 μL of virus lysate and dilute it 10-fold to 10,000-fold to obtain virus dilution solution for later use.

[0053] (2) Preparation of standard samples for standard curve Take 2×10 12 The plasmid standard of 1000 copies / mL was diluted with ultrapure water ddH2O into 6 gradients according to the proportion as the template of the standard 2×10 11 copies / mL, 2×10 10 copies / mL, 2×10 9 copies / mL, 2×10 8 copies / mL, 2×10 7 copies / mL, 2×10 6 copies / mL.

[0054] (3) Absolute quantitative qPCR 1) Take a 0.2mL PCR tube and prepare the following reaction system, with 3 replicates for each virus dilution. 2× qPCR Mix 10μL; 0.2μL each of forward and reverse primers; 5μL virus dilution; 4.2μL ddH2O; Amplification primers: Forward primer 5′- GGAACCCCTAGTGATGGAGTT-3′ (SEQ ID NO. 3); Reverse primer 5'- CGGCCTCAGTGAGCGA-3' (SEQ ID NO. 4); 2) qPCR amplification conditions Pre-denaturation: 95°C, 2 min; 40× cycles: 95°C, 15 s; 60°C, 60 s; 3) qPCR data processing: Virus titer = dilution factor * virus gene array copy number Virus yield = virus titer * virus volume.

[0055] The present invention adopts 3 plasmid systems to package pAAV2-WT and pAAV2 mutants respectively, using the suspension cell transient transfection method to package the virus, lysing the cells after 72 hours to harvest the virus, and purifying the virus by iodixanol density gradient centrifugation, and measuring the virus yield by QPCR. The experimental results are shown in Figure 1 As shown in the figure, it can be seen that the yields of different mutants vary greatly. The virus yields of AAV2-NE01 and AAV2-NE02 are 1.96 times and 1.20 times that of AAV2-WT, and the virus yields of the remaining 28 mutants are 0.04 to 1.26 times that of AAV2-WT. AAV2-NE01, AAV2-NE02 and mutants with a yield not less than 80% of the AAV2-WT yield were selected for in vivo infection ability testing in crab-eating monkeys.

[0056] 2. Test on infection effect of eye tissue in cynomolgus monkeys AAV2-WT and AAV2-mutant viruses were injected into cynomolgus monkeys, and 3 weeks later, the eyeballs were removed and frozen sections were prepared.

[0057] (1) Intravitreal injection The samples of Example 1 and the comparative example were diluted to 1E+12 vg / µL, and injected into the vitreous cavity of cynomolgus monkeys, with 100 µL injected into each eye (total virus amount was 1E+11 vg), and each virus was injected into one cynomolgus monkey. The specific operation steps are as follows: 1) Before injecting anesthesia, an ophthalmologist will perform an ocular examination (slit lamp) to observe the ocular surface. If any abnormality is found, it will be removed to ensure that there is no ocular lesion; 2) After the cynomolgus monkeys were anesthetized, tropicamide was instilled into the eyeball to dilate the pupil, and then tetracaine hydrochloride was instilled into the eyeball for surface anesthesia after the pupil was dilated; 3) Use a microsyringe to draw up an appropriate amount of virus for later use. Place the anesthetized crab-eating macaque on the operating table and give the eyes local anesthesia with lidocaine hydrochloride. Press the head with the left hand to make the eyeball protrude. Hold the microsyringe with the right hand and insert it into the vitreous cavity from the posterior edge of the cornea. The needle tip should first enter vertically and then tilt to slowly push the drug in. After the injection, slowly withdraw the needle.

[0058] (2) Sampling, frozen section preparation and fluorescence signal observation 3 weeks after virus injection, the cynomolgus monkeys were euthanized, the eyeballs were removed, frozen sections were prepared, and the expression of GFP fluorescence signals in various areas of the eye tissue was observed. The steps are as follows: 1) Dissect the fresh eyeball tissue and immediately fix it in tissue fixative for 24h-48h; 2) After taking the tissue out of the fixative, remove the surrounding tissue and excess tissue, cut it open with a sharp blade according to the experimental requirements, remove the lens and vitreous body inside, and dehydrate the trimmed eyeball tissue block in a 10% sucrose-20% sucrose-30% sucrose solution in a 4°C refrigerator; 3) Take out the dehydrated tissue, dry the surface water with filter paper, and place it in the embedding frame with the cut surface facing up. Add OCT embedding medium (avoid bubbles), put the embedding frame in a -20℃ refrigerator and freeze for half an hour, then transfer to a -80℃ refrigerator for storage; 4) Before slicing, take out the OCT embedded block from -80℃ and place it in the freezing section machine to equilibrate the temperature for 30 minutes, load the sample and slice, cut the slices according to the thickness required by the experiment, label the slices and store them at -20℃ for later use; 5) Add DAPI prepared in a certain proportion on the slices, incubate at room temperature in the dark for 3 minutes, place the slides in PBS and wash 3 times, 5 minutes each time, and seal the slides with anti-fluorescence quenching sealing agent; 6) Collect pictures before observation using a fluorescence microscope.

[0059] (3) Immunofluorescence staining After the frozen sections were labeled with Anti-Rhodopsin antibodies, the co-localization of GFP or Mcherry with visual cells was observed under a fluorescence microscope. The specific steps are as follows: 1) Take the frozen sections out of the -20℃ freezer, restore to room temperature, and dry. If the samples are not fixed, fix them with tissue fixative for 15 minutes and then rinse with running water; 2) Place the slides in the repair solution, boil them in a pressure cooker and purify them for 3 minutes. After cooling naturally, place the slides in PBS and wash them 3 times, 5 minutes each time. 3) Place the slices in 0.3% Triton X-100 solution, incubate at room temperature in the dark for 10 min, place the slides in PBS and wash three times, 5 min each time; 4) Add blocking serum to the tissue circle to evenly cover the tissue and block at room temperature for 60 minutes; 5) Gently shake off the blocking solution, add the primary antibody prepared in a certain ratio (Anti-Rhodopsin antibody abcam, ab221664, 1:500) on the slices, and incubate the slices flat in a humidified box at 4°C overnight or at room temperature for 1 hour; 6) Wash the slides in PBS for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (fluorescent label) of the same species as the primary antibody to cover the tissue and incubate at room temperature for 60 minutes. Wash the slides in PBS for 3 times, 5 minutes each time. Wash the slides in PBS for 3 times, 5 minutes each time; 7) Add DAPI prepared in a certain proportion on the slices, incubate at room temperature in the dark for 3 minutes, place the slides in PBS and wash 3 times, 5 minutes each time; 8) Use anti-fluorescence quenching sealing agent for sealing; 9) Observe the fluorescence signal under a fluorescence microscope.

[0060] 3 weeks after virus injection, samples were taken to make frozen sections, and the eyeball sections were stained with DAPI. The results were observed under a fluorescence microscope. Figure 2 and Figure 3 As shown, after intravitreal injection, AAV2-NE01-CAG-EGFP (green) and AAV2-NE02-CAG-Mcherry (red) viruses can well infect the cells of the NHP retina from the ganglion cell layer (GCL), inner nuclear layer (INL) to the outer nuclear layer (ONL) ( Figure 2 ) and trabecular meshwork area ( Figure 3 ), high infection positive rate, strong fluorescence intensity, AAV2-NE01 infection range and positive rate are slightly better than AAV2-NE02. AAV2-WT only infects a small amount of RGC layer cells, but has no infection on other layers of the NHP retina and trabecular meshwork, and other variants have no good infection effect. It is proved that the adeno-associated virus variants AAV2-NE01 and AAV2-NE02 prepared by the scheme of the present invention have significantly improved the ability to infect NHP eye tissues.

[0061] In order to further and more accurately locate the layers of retinal cells infected by AAV2-NE01 and AAV2-NE02, frozen sections of eye tissue were labeled and stained with Anti-Rhodopsin (antibody specific for the outer segment of visual cells). The results are shown in the figure. Figure 4 and Figure 5 As shown in the figure, it can be seen that the GFP signal and the mcherry signal overlap with the weaker part of the Anti-Rhodopsin signal, and the stronger part is closely connected. Rhodopsin is weakly expressed in the nuclei and inner segments of cone and rod cells, and strongly expressed in the outer segments of cone and rod cells, further proving that AAV2-NE01 and AAV2-NE02 have strong ability to penetrate the retina and can infect the nuclei and inner segments of photoreceptors.

[0062] The sequence alignment results of the adeno-associated virus variant capsid protein sequences AAV2-NE01 (SEQ ID NO.: 1) and AAV2-NE02 (SEQ ID NO.: 2) and the wild-type AAV2 capsid protein are shown in the figure below: Figure 6 The difference between the sequences of the adeno-associated virus variant capsid proteins AAV2-NE01 and AAV2-NE02 and the wild-type AAV2 capsid protein (protein ID: YP_680426.1) is that the AAVRFDGTERAA and RQYSDAVRAE polypeptide sequences are inserted into the amino acids 587 and 588 of the wild-type AAV2 virus capsid protein, respectively.

[0063] In summary, the adeno-associated virus variant prepared by the scheme of the present invention can significantly enhance the NHP retinal cells of various layers of the AAV2 virus compared with the wild type.

[0064] Amino acid sequence of adeno-associated virus variant AAV2-NE01 capsid protein Cap, SEQ ID NO.1: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHF SPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLID QYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAVRFDGTERAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL。

[0065] Amino acid sequence of the capsid protein Cap of adeno-associated virus variant AAV2-NE02, SEQ ID NO.2: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN QYSDAVRAE RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL。

[0066] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A capsid protein mutant that improves the ability of AAV to infect eye tissues, characterized in that: The polypeptide is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the AAV capsid protein, and the insertion site is homologous to the amino acids 587 and 588 of the wild-type AAV2 virus capsid protein; preferably, the polypeptide is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE into the capsid protein of the AAV2 variant, and the insertion site is homologous to the amino acids 587 and 588 of the wild-type AAV2 virus capsid protein.

2. The capsid protein mutant according to claim 1, characterized in that It is obtained by inserting the polypeptide AAVRFDGTERAA or RQYSDAVRAE between amino acids 587 and 588 of the wild-type AAV2 capsid protein.

3. A gene encoding the capsid protein mutant according to claim 1 or 2.

4. An expression vector, characterized in that: It expresses the capsid protein mutant described in claim 1 or 2, or has the gene described in claim 3 inserted therein.

5. The expression vector according to claim 4, characterized in that The expression vector is a plasmid or a recombinant AAV vector.

6. The expression vector according to claim 5, characterized in that The AAV is selected from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10 and variants thereof.

7. The expression vector according to claim 5, characterized in that Also included are genes encoding functional gene products.

8. An AAV virus particle, characterized in that The outer shell is formed by assembling capsid proteins VP1, VP2 and VP3 formed by the capsid protein mutants described in claim 1 or 2, or by assembling capsid proteins VP1, VP2 and VP3 encoded by the gene described in claim 3.

9. A composition, characterized in that It comprises the expression vector according to any one of claims 4 to 7, or the AAV virus particle according to claim 8.

10. Use of the composition according to claim 9, characterized in that: The applications include: Preparation of gene therapy formulations targeting ocular tissues; Construct transgenic animal models of eye tissue diseases; Preparation of drugs or transgenic vectors targeted to ocular tissues.

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